Development of an improved blood-stage malaria vaccine targeting the essential RH5-CyRPA-RIPR invasion complex

Reticulocyte-binding protein homologue 5 (RH5), a leading blood-stage Plasmodium falciparum malaria vaccine target, interacts with cysteine-rich protective antigen (CyRPA) and RH5-interacting protein (RIPR) to form an essential heterotrimeric “RCR-complex”. We investigate whether RCR-complex vaccination can improve upon RH5 alone. Using monoclonal antibodies (mAbs) we show that parasite growth-inhibitory epitopes on each antigen are surface-exposed on the RCR-complex and that mAb pairs targeting different antigens can function additively or synergistically. However, immunisation of female rats with the RCR-complex fails to outperform RH5 alone due to immuno-dominance of RIPR coupled with inferior potency of anti-RIPR polyclonal IgG. We identify that all growth-inhibitory antibody epitopes of RIPR cluster within the C-terminal EGF-like domains and that a fusion of these domains to CyRPA, called “R78C”, combined with RH5, improves the level of in vitro parasite growth inhibition compared to RH5 alone. These preclinical data justify the advancement of the RH5.1 + R78C/Matrix-M™ vaccine candidate to Phase 1 clinical trial.

Full GIA dilution curves of pairwise mAb combinations targeting the RCR-complex.Black: Titrated mAb alone.Red: predicted Bliss additivity GIA for combination of the two mAbs with one held constant at ~30 % GIA.Blue: measured GIA data of the combination of the titrated mAb plus held concentration of the other mAb.Individual points are the mean of a triplicate measurement with error bars indicating the standard deviation.Source data are provided as a Source Data file.Figure S3 -Rat immunisation study with full-length RCR-complex antigen combinations.Wistar rats were immunised intramuscularly with soluble protein vaccines formulated in Matrix-M™ adjuvant on days 0, 28 and 56.Tail bleeds were taken on day -2 (pre-immunisation) and day 42 (post-second dose), followed by terminal bleed on day 70 (post-third dose).(A) Serum IgG ELISA data shown for samples taken on days 42 and 70 against full-length RH5 (red), CyRPA (blue), and RIPR (green) reported in µg/mL.Individual and median group responses are shown.Dotted line corresponds to median antigen-specific IgG response from the relevant group of single antigen immunised animals.(B) Single-cycle GIA assay using P. falciparum clone 3D7.Total IgG was purified from day 70 serum samples and titrated in the GIA assay.Each line represents an individual animal, and each point represents the mean of three technical replicates.GIA at 1 mg/mL total purified IgG was interpolated for each animal.(C) Data from (B) replotted against total antigen-specific IgG concentration in µg/mL as measured by ELISA in each purified total IgG sample.Each dataset was fitted with a Richard's five-parameter dose-response curve with no constraints to ascertain the EC50.Doses of each antigen in µg used for vaccination are specified in brackets in the title of each graph.Source data are provided as a Source Data file.Wistar rats were immunised intramuscularly with soluble protein vaccines formulated in 25 µg Matrix-M™ adjuvant on days 0, 28 and 56.Tail bleeds were taken on day -2 (pre-immunisation), day 14 (post-first dose) and day 42 (post-second dose), followed by terminal bleed on day 70 (post-third dose).(A) Serum IgG ELISA data shown for samples taken on days 14, 42 and 70 against full-length RH5 (red), CyRPA (blue), and RIPR (green) reported in µg/mL.Individual and median group responses are shown.Dotted line corresponds to median antigen-specific IgG response from the relevant group of single antigen immunised animals where applicable.Data are spread over two graphs to show separate immunisation cohorts.(B) Single-cycle GIA assay using P. falciparum clone 3D7.Total IgG was purified from day 70 serum samples and titrated in the GIA assay.Each line represents an individual animal, and each point represents the mean of three technical replicates.GIA at 1 mg/mL total purified IgG was interpolated for each animal.(C) Data from (B) replotted against total antigenspecific IgG concentration in µg/mL as measured by ELISA in each purified total IgG sample.Each dataset was fitted with a Richard's five-parameter dose-response curve with no constraints to ascertain the EC50.Doses in µg of each antigen used for vaccination are specified in brackets in the title on each graph.Source data are provided as a Source Data file.

Figure S2 -
Figure S2 -Analysis of inter-antigen synergistic GIA of monoclonal antibodies targeting the RCRcomplex.Full GIA dilution curves of pairwise mAb combinations targeting the RCR-complex.Black: Titrated mAb alone.Red: predicted Bliss additivity GIA for combination of the two mAbs with one held constant at ~30 % GIA.Blue: measured GIA data of the combination of the titrated mAb plus held concentration of the other mAb.Individual points are the mean of a triplicate measurement with error bars indicating the standard deviation.Source data are provided as a Source Data file.

Figure S5 -
Figure S5 -Production of RIPR protein fragments, antigen-reversal GIA assays and anti-RIPR mAb epitope mapping.(A)Schematic of full-length RIPR protein (RIPR FL) with the design of each RIPR protein fragment labelled with first and last amino acid.EGF domains shown as blue circles.Chen3 and Chen1/2 were previously reported as "Ripr3" and "Ripr1/2" by Chen et al.3 (B) Reducing SDS-PAGE of RIPR FL and RIPR fragments fused to the monoFc prior to cleavage by TEV protease.CTD = C-terminal domain.MonoFc-GFP fusion protein produced as a control.(C) Single cycle GIA assay using P. falciparum clone 3D7 of purified IgG from rabbits immunised with full-length RIPR (blue, N=6) or from a preimmune serum negative control (grey, N=1).Each dataset fitted with a Richard's five-parameter dose-response curve with no constraints.Mean of triplicate wells and SD shown.(D) Antigen reversal single cycle GIA assay using P. falciparum clone 3D7 and pooled anti-RIPR IgG from (C).Total purified IgG was held at 3 mg/mL and the indicated RIPR proteins were included at1 µM final concentration unless otherwise indicated.Blue bar = anti-RIPR IgG alone; white bars = anti-RIPR IgG + indicated protein; grey bars = indicated protein alone; green bars = anti-RH5 polyclonal IgG control (pAb) with (+) or without (-) addition of RIPR EGF(7-8) protein.Mean and SD of triplicate wells shown.Significance determined by Kruskal-Wallis test with Dunn's multiple comparison post-test versus anti-RIPR FL IgG alone; * p<0.05, ** p<0.01.(E) Antigen reversal single cycle GIA assay using P. falciparum clone 3D7 and pooled anti-RIPR FL IgG from (C).Total purified IgG was held at 3 mg/mL and the indicated RIPR EGF proteins were titrated from 20 µM to 0.31 µM final concentration.Coloured bars = anti-RIPR IgG + indicated protein; grey bars = indicated protein alone; blue bars = controls: Control 1 = 3 mg/mL anti-RIPR IgG alone.Control 2 = 20 µM RIPR EGF(5-6) or RIPR EGF(7-8) alone.Control 3 = anti-RH5 IgG at 3 mg/mL and Control 4 = 20 µM indicated RIPR EGF protein with anti-RH5 IgG at 3 mg/mL.Control 5 = RIPR EGF(5-6) and RIPR EGF(7-8) proteins mixed at 20 µM and tested alone.All bars are mean and error bars are SD, N=3 except for EGF(5-8) where N=6.(F) Identification of anti-RIPR mAb binding regions by dot blot using full-length RIPR and indicated RIPR protein fragments.A subset of mAbs were also tested against denatured full-length RIPR protein.(G) Anti-RIPR mAbs RP.016 and RP.017 bound denatured RIPR and Nhalf by dot blot and were then screened on an overlapping peptide ELISA covering amino acids 21-247 and 364-648; both mAbs bind to peptide 1 (IDLIEGIFYEKNEIDKLTFS).Representative graph of two repeats shown.Source data are provided as a Source Data file.

tr o l 4 Figure S6 -Figure S7 -Figure S8 -
Figure S6 -Investigation of RIPR interactions with SEMA7A and PTRAMP-CSS.(A) SPR sensorgrams show multi-cycle kinetics of CyRPA (left) or SEMA7A (right) binding to RIPR coated on a CM5 chip.Report points of the equilibrium binding levels of the 9-step 2-fold dilutions for CyRPA binding to RIPR are fitted to the equilibrium or steady-state binding model for KD determination (middle).No RIPR binding detected for SEMA7A.(B) SPR sensorgrams show multicycle kinetics of MTRAP (left) or RIPR (right) binding to SEMA7A coated on a CM5 chip.Report points of the equilibrium binding levels of the 9-step 2-fold dilutions for MTRAP binding to SEMA7A are fitted to the equilibrium or steady-state binding model for KD determination (middle).No SEMA7A binding detected for RIPR.(C) SDS-PAGE gel showing the recombinant PTRAMP-CSS heterodimer run under non-reducing (N) and reducing (R) conditions.Arrows indicate PTRAMP-CSS heterodimer (expected mass 62 kDa, PTRAMP and CSS 31 kDa each), confirmed by mass spectrometry analysis (data not shown).Higher band is an unidentified contaminant from the baculovirus expression system.(D) Western blot of gel shown in (C) using streptavidin-AP to detect biotin added to the BAP tag by BirA.(E) SPR sensorgrams show multi-cycle kinetics of RIPR binding to PTRAMP-CSS heterodimer coated on a CM5 chip.Curves show a 5-step 2-fold dilution series for RIPR binding.The equilibrium or steady-state binding model was used for KD determination.(F) No binding of RIPR EGF(5-8) to the PTRAMP-CSS heterodimer could be detected by the same method as (E).Source data are provided as a Source Data file.

Figure S4 -Calibration free concentration analysis (CFCA) for rat IgG and rat immunisation study with the single full-length RH5, CyRPA and RIPR antigens.
J) re-plotted against the appropriate anti-antigen specific IgG content of each sample as measured by ELISA in µg/mL.N=12 animals per antigen, each dataset fitted with a Richard's fiveparameter dose-response curve with no constraints.The dashed line shows 0 % GIA and dotted line shows 50 % GIA for comparison.(N) Summary table of interpolated GIA EC50 results for each immunisation antigen.Source data are provided as a Source Data file.

Table S1 -Details of all monoclonal antibodies used in this study.
NA = Not applicable.NT = Not tested.RH5 epitope bin refers to colour coding reported by Alanine et al 1 .

Table S2 -Summary statistics for rat immunisation study using full-length RCR antigens.
Summary statistics for ELISA data shown in Figure 4A-C.Sections A-C of the table correspond to Figure 4A-C.Significance determined by one-way ANOVA with Dunnett's multiple comparisons test versus the single antigen only group as indicated, * p<0.05, *** p<0.0001, **** p<0.0001, ns = not significant. .Oneway ANOVA on log-transformed data with Dunnett's multiple comparison test †Performed on CyRPA data from panel C ‡ Performed on RIPR data from panel E

Table S3 -Summary statistics for rat immunisation study using R58C and R78C antigens.
Summary statistics for ELISA data shown in Figure 6A-F.Sections A-F of the table correspond to Figure 6A-F.Significance determined by one-way ANOVA of data with Dunnett's multiple comparisons test versus the single antigen only group as indicated, * p<0.05, ** p<0.001, **** p<0.0001, ns = not significant.